Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca2+ concentration ((Ca2+)i), the present study aimed to determine the changes in Ca2+ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca2+ store of freshly prepared milk cells was estimated from the elevation of (Ca2+)i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca2+ store in milk cells after intracellular Ca2+ depletion by Bapta-AM followed by spiking with 2.5 mM Ca2+ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca2+ store in milk cells was much less than blood PMNs. The production of superoxide anion (O2−) in vitro in response to (Ca2+)i-dependent or (Ca2+)i-independent modulators was used to evaluate the relevance of altered Ca2+ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O2− as blood PMNs when treated with ionomycin. However, the amount of O2− produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O2− production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca2+ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O2–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca2+, but decreased O2− production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca2+ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.